During spermiogenesis, spermatids acquire the acrosome and flagellum, condense their chromatin and become spermatozoa. Precise expression of spermatid differentiation markers is critical for proper formation of sperm. The overall goal of our research is to understand the mechanisms of transcriptional regulation of spermiogenesis. The gene encoding the acrosomal protein SP-10 serves as an experimental model. Promoter analysis showed that the proximal promoter of the SP-10 gene was sufficient for transcriptional activation in spermatids. Surprisingly, the same promoter caused transcriptional repression in all other cells by acting as an insulator. The central hypothesis is that coordinated interplay of testis-specific transcriptional activators and ubiquitously expressed transcriptional repressors dictates the precise stage- and cell-specific expression of spermatid differentiation markers and that the germ cell-specific genes retain the necessary cis-elements in the proximal promoters. This proposal will test the hypothesis that in vivo, specific cis elements in the SP-10 proximal promoter mediate the enhancer function in spermatids and insulator / repressor function in all other cells, and that the cognate transcriptional activators and repressors alternate promoter occupancy to induce ON and OFF states of SP-10 gene transcription, respectively. The study will focus on the -186/+28 SP-10 promoter, which showed both enhancer and insulator activities, and characterize transcription factors mediating these functions. TNP47, a 47kD testis nuclear protein, which specifically binds the -186/-148 region in vitro, will be cloned by DNA affinity method and its role in SP-10 transcription determined (Aim 1). Whether TDP43 and PURalpha, transcriptional repressors cloned using the -186/-148 DNA, mediate the SP-10 insulator function will be determined by cotransfections and RNAi, their promoter occupancy in vivo will be determined by chromatin IP (Aim 2). The hypothesis that the coordinated interplay of TNP47, TDP43 and PURalpha is sufficient to ensure spermatid-specific transcription will be tested in transgenic mice using the -186/-148 regulatory promoter in the context of its native (SP-10) as well as heterologous core promoters (Aim 3). The results will enumerate molecular mechanisms underlying spermiogenesis. The study is relevant to male reproductive health as leads can be applied to develop novel male contraceptives. The SP-10 promoter will be useful for gene therapy to treat infertility or germ cell defects.